Team:UNIPV-Pavia/Calendar/July/settimana3

From 2010.igem.org

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Trasformation of ligations in:
Trasformation of ligations in:
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*I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
*I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)

Revision as of 13:50, 26 July 2010

JULY: WEEK 3



July, 12th

Phasins PhaP1 and PhaP2 were sequenced, but none of them has a clear chromatogram, so we decided to sequence the PCR results.

Gel run/cut of phasins amplified via PCR

Phasins were gel run/cut and the samples were prepared to be sequenced.

Inoculum of

  • <partinfo>BBa_J23101</partinfo>
  • <partinfo>BBa_J23105</partinfo>
  • <partinfo>BBa_J23106</partinfo>

for tomorrow ligations.

July, 13th

LB+Amp was prepared and phasins sample were sent to be sequenced.

For cultures grown OverNight at 37°C, 220 rpm were MiniPrep was performed:

<partinfo>BBa_J23101</partinfo> XXX ng/ul
<partinfo>BBa_J23105</partinfo> XXX ng/ul
<partinfo>BBa_J23106</partinfo> XXX ng/ul

Other plasmids were retrieved from our freezer:

  • <partinfo>BBa_J23118</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23110</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23116</partinfo> (already digested SpeI-PstI)
  • I6-2 (already digested XbaI-PstI)
  • 4C5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I7-3 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I8-5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I10-1 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I3-1 (MiniPrep performed, it will be digested XbaI-PstI)


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
4C5 (x2) Vector 25 3,6 16,9 1 EcoRI 1 PstI 2,5
I7-3 Insert 25 14 6,5 1 EcoRI 1 PstI 2,5
I8-5 Insert 25 12,7 7,8 1 EcoRI 1 PstI 2,5
I10-1 Insert 25 15,3 5,2 1 EcoRI 1 PstI 2,5
I3-1 (x2) Vector 25 5,8 14,7 1 XbaI 1 PstI 2,5
<partinfo>BBa_J23105</partinfo> Insert 25 15,9 4,6 1SpeI 1 PstI 2,5
<partinfo>BBa_J23106</partinfo> Insert 25 13 7,5 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23101</partinfo> Insert 25 10,4 10,1 1 SpeI 1 PstI 2,5

Digestions were incubated at 37°C for 3hours, then gel run/cut.

Gel was prepared for electrophoresis: 150ml TBE + 1,5 g Agarose + 3ul EtBr.

Ligations were all performed 1:5 (1ul vector + 5ul insert):

  • I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
  • I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
  • I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
  • I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
  • I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
  • I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
  • I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
  • I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
  • I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
  • I74C5= I7 (E-P) + 4C5 (E-P)
  • I84C5= I8 (E-P) + 4C5 (E-P)
  • I104C5= I10 (E-P) + 4C5 (E-P)

July, 14th

Trasformation of ligations in:

  • I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)

TOP10

  • I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)

TOP10

  • I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)

TOP10

  • I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)

DH5alpha

  • I74C5= I7 (E-P) + 4C5 (E-P)

TOP10

  • I84C5= I8 (E-P) + 4C5 (E-P)

TOP10

  • I104C5= I10 (E-P) + 4C5 (E-P)

TOP10


Quality control for HELPER and CRIM plasmids

July, 15th

Quality control for pBHR68 and CRIM plasmids

July, 16th

Screening ligations I11-I16
Screening ligations I17-I19, I7/I8/I10-4C5


Since it's impossible to isolate the CRIM plasmid <partinfo>BBa_J72007</partinfo> we created a pseudo CRIM plasmid to check that not pir strains aren't able to propagate it. We called it "THE RING" (RING) and we built it from our I5 (Cm resistance - <partinfo>BBa_P1004</partinfo> - + TT - <partinfo>BBa_B0015</partinfo> - + R6K ori - <partinfo>BBa_J61001</partinfo>) in <partinfo>pSB1A2</partinfo>:

  • Digestion of I5 XbaI-PstI
  • Gel extraction
  • Quantification with Nanodrop (15ng/ul)
  • Ligation of X and P sites each other (used 2ul of DNA)


Calendar

July

week 1

week 2

week 3

week 4

week 5